SummaryMicroRNAs (miRNAs) are instrumental to many aspects of immunity, including various levels of T-cell immunity. Over the last decade, crucial immune functions were shown to be regulated by specific miRNAs. These 'immuno-miRs' regulate generic cell biological processes in T cells, such as proliferation and apoptosis, as well as a number of T-cell-specific features that are fundamental to the development, differentiation and function of T cells. In this review, we give an overview of the current literature with respect to the role of miRNAs at various stages of T-cell development, maturation, differentiation, activation and ageing. Little is known about the involvement of miRNAs in thymic T-cell development, although miR-181a and miR-150 have been implicated herein. In contrast, several broadly expressed miRNAs including miR-21, miR-155 and miR-17~92, have now been shown to regulate T-cell activation. Other miRNAs, including miR146a, show a more T-cell-subset-specific expression pattern and are involved in the regulation of processes unique to that specific T-cell subset. Importantly, differences in the miRNA target gene repertoires of different T-cell subsets allow similar miRNAs to control different T-cell-subsetspecific functions. Interestingly, several of the here described immuno-miRs have also been implicated in T-cell ageing and there are clear indications for causal involvement of miRNAs in immunosenescence. It is concluded that immuno-miRs have a dynamic regulatory role in many aspects of T-cell differentiation, activation, function and ageing. An important notion when studying miRNAs in relation to T-cell biology is that specific immuno-miRs may have quite unrelated functions in closely related T-cell subsets.

Tamoxifen (TAM) inducible Cre recombinase system is an essential tool to study gene function when early ablation or overexpression can cause developmental defects or embryonic lethality. However, there remains a lack of consensus on the optimal route and dosage of TAM administration in vivo. Here, we assessed dosage and delivery of TAM for activation of Cre in immune cell subsets assessed longitudinally and spatially using transgenic mice with ubiquitously expressed Cre/ER and the Cre-inducible fluorescent reporter YFP. After comparing two TAM delivery methods (intraperitoneal versus oral gavage) and different doses, we found that 3 mg of TAM administered orally for five consecutive days provides maximal reporter induction with minimal adverse effects in vivo. Serum levels of TAM peaked 1 week after initiating treatment then slowly decreased, regardless of dosing and delivery methods. TAM concentration in specific tissues (liver, spleen, lymph nodes, and thymus) was also dependent on delivery method and dose. Cre induction was highest in myeloid cells and B cells and substantially lower in T cells, and double-positive thymocytes had a notably higher response to TAM. In addition to establishing optimal dose and administration of TAM, our study reveals a disparate activity of Cre in different cell immune populations when using Cre/ER models.

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T-cell activation affects microRNA (miRNA) expression in T-cell subsets. However, little is known about the kinetics of miRNA regulation and possible differences between CD4 and CD8 T cells. In this study we set out to analyze the kinetics of activation-induced expression regulation of twelve pre-selected miRNAs. The dynamics of the expression of these miRNAs was studied in sorted CD4 and CD8 CD45RO- T cells of healthy individuals stimulated with αCD3/αCD28 antibodies. Analysis of miRNA levels at day 3, 5, 7 and 10 showed significant activation-induced changes in expression levels of all twelve miRNAs. Expression levels of nine miRNAs, including miR-21, miR-146a and miR-155, were induced following activation, whereas expression of three miRNAs, including miR-31, were decreased following activation. The expression changes of miR-18a and miR-155 was relatively early, at day 3, whereas expression of miR-451, miR-21 and miR-146a was evident at day 5, 7 and 10, respectively. Four miRNAs showed a differential regulation between CD4 and CD8 T cells. Induction of miR-18a and miR-21 was more pronounced and occurred earlier in CD4 T cells compared to CD8 T cells. Downregulation of miR-223 and miR-451 was also more pronounced in CD4 T cells compared to CD8 T cells. In conclusion, we show a complex pattern of miRNA expression regulation upon T-cell activation with early and late as well as CD4 and CD8 T-cell specific changes. These differences might be the result of differences in kinetics and efficiency of CD4 and CD8 T cells in response to antigen priming.

The probability of atherosclerotic plaque rupture and its thrombotic sequelae are thought to be increased at sites of macrophage accumulation. Folate receptor b (FR-b) is present on activated macrophages but not on quiescent macrophages or other immune cells. By conjugating the ligand folate with a fluorescent contrast agent, fluorescein isothiocyanate (FITC), we aimed to explore the potential role of FR-b fluorescence imaging in the distinction of vulnerable sites from more stable regions. Methods: Carotid specimens were taken from 20 patients and incubated with folate-FITC for 30 min. Ex vivo fluorescence imaging was performed to determine the exact location of folate-FITC uptake. Sections displaying regions of high uptake (determined as hot spots) were compared with sections showing low uptake (cold spots) through immunohistochemistry and real-time quantitative reverse-transcription polymerase chain reaction for FR-b. Results: Hot spots showed significantly higher folate-FITC uptake than cold spots (P , 0.001). Hot spots tended to contain more macrophages and areas of hypoxia than cold spots. A positive correlation between messenger RNA levels of CD68 (marker for macrophages), FR-b (r 5 0.53, P 5 0.045), and hypoxia-inducible factor-1a expression (marker for intraplaque hypoxia; r 5 0.55, P 5 0.034) was found. Conclusion: Compared with areas with low folate-FITC uptake, areas of high folate-FITC uptake within human atherosclerotic plaques had an increased number of activated macrophages and higher areas of hypoxia. These characteristics of vulnerability imply that molecular imaging of FR-b through folate conjugates might be a good indicator for plaque vulnerability in future noninvasive imaging studies.

Type 1 diabetes (T1D) arises from a failure to maintain tolerance to specific β-cell antigens. Antigen-specific immunotherapy (ASIT) aims to reestablish immune tolerance through the supply of pertinent antigens to specific cell types or environments that are suitable for eliciting tolerogenic responses. However, antigen-presenting cells (APCs) in T1D patients and in animal models of T1D are affected by a number of alterations, some due to genetic polymorphism. Combination of these alterations, impacting the number, phenotype, and function of APC subsets, may account for both the underlying tolerance deficiency and for the limited efficacy of ASITs so far. In this comprehensive review, we examine different aspects of APC function that are pertinent to tolerance induction and summarize how they are altered in the context of T1D. We attempt to reconcile 25 years of studies on this topic, highlighting genetic, phenotypic, and functional features that are common or distinct between humans and animal models. Finally, we discuss the implications of these defects and the challenges they might pose for the use of ASITs to treat T1D. Better understanding of these APC alterations will help us design more efficient ways to induce tolerance.

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Thymic selection constitutes the first checkpoint in T-cell development to purge autoreactive T cells. Most of our understanding of this process comes from animal models because of the challenges of studying thymopoiesis and how T cell receptor (TCR) specificity impacts thymocyte phenotype in humans. We developed a humanized mouse model involving the introduction of autoreactive TCRs and cognate autoantigens that enables the analysis of selection of human T cells in human thymic tissue in vivo. Here, we describe the thymic development of MART1-specific autoreactive CD8+ T cells that normally escape deletion and how their phenotype and survival are affected by introduction of the missing epitope in the hematopoietic lineage. Expression of the epitope in a fraction of hematopoietic cells, including all major types of antigen-presenting cells (APCs), led to profound yet incomplete deletion of these T cells. Upregulation of PD-1 upon antigen encounter occurred through the different stages of thymocyte development. PD-1 and CCR7 expression were mutually exclusive in both transgenic and non-transgenic thymocytes, challenging the view that CCR7 is necessary for negative selection in humans. In the presence of antigen, MART1-reactive T cells down-regulated TCR, CD3, CD8, and CD4 in the thymus and periphery. Moreover, expression of secondary TCRs influences MHC class I-restricted T cells to develop as CD4+, particularly regulatory T cells. This new model constitutes a valuable tool to better understand the development of autoreactive T cells identified in different human autoimmune diseases and the role of different APC subsets in their selection.